Stable and targeted delivery of small interfering RNA (siRNA) to diseased sites has been a key obstacle for clinical translation of siRNA based therapeutics. Transport of siRNA to cytoplasm for effecting gene therapy is another obstacle. Antibodies that bind to specific biomarkers in the cell and also aide in retaining the stability of siRNA provide a partial solution for stable delivery, however they fail to deliver siRNA effectively to cytoplasm of the infected cells.
Small interfering RNA (siRNA) is a promising therapeutic route for several infected cell diseases. In the case of cancer, it is known that standalone siRNA does not cause cytotoxicity to cells to the extent that infected cells are completely eliminated. A knockdown of an oncogene merely silences the gene of interest, and consequently facilitates the affected cancer cells to adapt and adopt a different pathway of survival. For definitive cytotoxic action, a molecule complementing the siRNA for inducing inclusive toxicity needs to be delivered simultaneously and at appropriate proportions to the infected cells where the siRNA is delivered. Conversion of an un-druggable ailment to a drug responsive one through simultaneous delivery of siRNA and cytotoxin to infected cells will pave way for using siRNA therapy for drug-resistant diseases. Indeed, the efficacy would be higher when this complementarily paired system is targeted towards specific biomarkers present on the surface of the infected cells.
In accordance to numerous publications on the delivery of siRNA, the essential parameters that govern effective delivery of siRNA can be categorized into (1) stability of siRNA, (2) targeting the siRNA, (3) effective gene silencing, and (4) cytotoxicity and off-target effects. For stable siRNA delivery, Mirkin and co-workers reported AuNP functionalized with oligoethylene glycol-siRNA. The study showed that the OEG performed two essential functions in combination with gold nanoparticles; namely, polyvalency for inducing proton sponge effect, and relatively higher stability of siRNA in serum. The conjugate showed specific and higher knockdown of luciferase in HeLa cells compared to transfection with lipofectamine. Subsequent induction of cytotoxins and cellular response was not a part of the study. For targeting siRNA to specific biomarkers, Cuellar et al. studied siRNA-antibody conjugates (using THIOMAB) with various sets of antibodies and cell lines for targeted gene silencing.
The authors reported that, although the conjugates were very well defined with high targeting potential, the gene silencing aspect was not very effective for several cases. The reason for ineffective gene silencing was due to a relatively low proton-sponge effect for endosomal escape of siRNA. Again, subsequent cytotoxicity related studies were not a part of the work.
Non-Small Cell Lung Cancer (NSCLC) NSCLC is diagnosed in an estimated 220,000 patients each year with five-year overall survival rates of 16 percent.
A recent report confirmed that 16 percent of NSCLC patients carry oncogenic KRAS mutation. A potent drug targeted against KRAS mutation has not yet been developed and the objective response rate with the current standard of care is just three percent. An earlier report had suggested siRNA therapy renders the undruggable KRAS mutant cells to become susceptible to Tyrosine Kinase Inhibitors (TKI). Short interfering RNA (siRNA) is a well-known approach for effecting gene therapy to provide subsequent sensitization towards complementary therapeutic agents. However, stable delivery of siRNA is a significant challenge due to its high degradation rate in the presence of serum proteins and enzymes.
To overcome this challenge, several nanoparticle-based carrier systems have been attempted and those include retroviral vectors, liposomes, polymeric, and metallic nanoparticles. In these reported studies the physicochemical and surface properties of the particle were modified for delivering the siRNA to cytoplasm of the infected cells. Unfortunately, these nanoparticles suffer from serious limitations such as stability issues during synthesis, premature release in serum, inefficient endosomal escape, and interferon response. Importantly, oncogene knockdown alone has less impact on the cancer cell apoptosis since the cells tend to adopt another effector pathway for survival. Therefore, a need for complementary drug for initiating the apoptosis post knockdown is needed. Indeed, drugging cells separately and exogenously post oncogene knockdown has been reported earlier. A combined delivery system wherein, co-delivery of a drug along with siRNA to impede growth and survival of the cell has also been attempted. The relevance of the combined delivery is to ensure the complementary drug enters the same cells that are affected by siRNA at a predetermined appropriate proportion and time for causing cellular apoptosis. However, incorporation of siRNA (with minimal degradation) with a drug and a biomarker-targeting antibody into a single platform is synthetically challenging. Thus, stable and targeted delivery with concomitant cytotoxic action to cancer cells continues to be at early exploratory stages.
Significant efforts have been made to understand the downstream effect of oncogene knockdown mediated via siRNA. Cancer cells have several parallel working pathways, with one primary effector pathway coupled to several parallel effector pathways. The parallel pathways remain dormant until the working pathway is disrupted. Change in the protein expression levels upon knock down of oncogene present in the primary pathway results in change of downstream protein and gene expression levels regulated by complex cellular mechanism.
This mode of intra-cellular functioning adaptation evolves to drug resistance within cancer cells that are previously responding to therapy. On the other hand, KRAS mutant adenocarcinoma of NSCLC remains undruggable. While mutations occur at variation position of KRAS, oncogenic effect at codon 12 (Glycine-12 to Cysteine, G12C) of KRAS is the most commonly occurring mutation and yet to receive a dedicated drug. Although, in recent times, few attempts have been made for targeting G12C mutation through a small molecule inhibitor, RNAi therapy is emerging as a promising tool that could be applied across all types of mutations supplemented with currently approved drugs.
The present inventors have determined that depending on siRNA delivery through physical mixtures, electrostatic interaction of carrier vehicle and siRNA is not very effective. The low effectiveness can be attributed to the lack of definitive structural properties, and also possibly due to agglomeration of particles.
To date, no drug has been discovered which can inhibit the mutant KRAS for effecting therapy. Also, kinetics involved in the RAS pathway is highly complex and interlinked with several other intracellular pathways to assist in cell proliferation. Effect of cytotoxicity induction post oncogene knockdown was reported by Sunaga et al. for KRAS mutant NSCLC. In their report, un-druggable lung adenocarcinoma with K-Ras mutation showed sensitivity to a tyrosine kinase inhibitor (gefitinib) after knocking down K-Ras oncogene with retroviral vector carrying siRNA. The report is also one of the few wherein it was determined that post-knockdown, the NSCLC cells adopted an alternate downstream pathway for survival. The downstream Ras pathway upon knock-down of KRAS mutant gene decreased the protein level expression of the Ras pathway downstream protein pMEK, and also affected pAKT. The protein down-regulation effected an increase in pEGFR which is absent in the otherwise untreated cells, suggesting an alternate route of cellular mechanism. However, two main challenges limit the translation of retroviral vector strategy for the delivery of siRNA. Firstly, a retroviral vector carrying siRNA and functionalized with a targeting agent such as an antibody for targeting specific biomarkers is a synthetic challenge and has not been reported to this date to our knowledge. Secondly, ensuring the cytotoxin (such as for example, gefitinib) internalizes within all the cells altered by siRNA at appropriate relative proportions is difficult. An off-target interferon response due to retroviral vector might also create an issue for clinical translation.
Another cancer treatment target is AXL receptor which is overexpressed in various types of cancers. The AXL overexpression by some cancer cells has been reported in literature as a cause of cancer cells resistance to EGFR-targeting therapy. AXL (TAM receptor tyrosine kinase family; 140 kDa) is linked to cancer proliferation, migration and cause the subsequent resistance to small molecule tyrosine kinase inhibitors. AXL is also known to affect the PI3/Akt signaling pathways and induce a mutation in EGFR as a tyrosine kinase switch.